The overall objective is to determine the molecular and cellular mechanisms for insulin resistant glucose uptake in vivo in obesity and diabetes, with the long-term goal of developing novel strategies to reduce insulin resistance. Specific Aim 1 is to determine the relative importance of Glut4 in skeletal muscle and in adipocytes in the regulation of whole body glucose homeostasis and body composition. Transgenic mice will be generated in which Glut4 is "knocked out" selectively from adipose tissue or, separately, from skeletal muscle using Cre-recombinase loxP mediated gene targeting. These mice will be characterized in vivo for glucose homeostasis, insulin sensitivity, plasma lipid profiles, growth curves, body composition, food intake and plasma leptin and TNF levels. Obesity and insulin resistance will be included with high fat feeding and gold-thioglucose to determine whether mice with Glut4 functionally-ablated in adipose tissue are protected against developing obesity. If the tissue-specific Glut4 ablated mice are not diabetic, their susceptibility to develop severe insulin resistance or diabetes will be determined with high fat feeding and gold thioglucose-induced obesity. To investigate the mechanisms for in vivo effects, glucose transport and metabolism will be assessed in adipocytes and muscle in vitro. Leptin and TNF expression and secretion from adipocytes of adipose-specific-Glut4-ablation mice will be measured. Specific Aim 2 is to elucidate how activation of PI3 kinase is involved in Glut4 translocation by determining whether the subcellular localization of PI3 kinase activity is important. The feasibility of targeting PI3 kinase activity will be determined with fusion proteins. Constitutively active PI3 kinase will be overexpressed, with and without targeting it to intracellular membranes in insulin-responsive cultured adipocytes. Gene expression will be rapidly induced in fully differentiated 3T3-L1 adipocytes using adenovirus mediated gene delivery or tetracycline inducible gene expression. The effects of expressing activated PI3 kinase in intracellular membranes or diffusely in the cell on Glut4 translocation will be assessed. In future studies, inducible expression of kinase in skeletal muscle of transgenic mice will be used to determine whether activated PI3 kinase can overcome the Glut4 translocation/fusion/activation block associated with obesity and diabetes.